Journal of Soils and Sediments (2019) 19:2114–2125 https://doi.org/10.1007/s11368-018-2188-8

SEDIMENTS, SEC 4 • SEDIMENT-ECOLOGY INTERACTIONS • RESEARCH ARTICLE

Abundance, contribution, and possible driver of -oxidizing (AOA) in various types of aquatic

Weidong Wang1 & Weiyue Liu1,2 & Shanyun Wang1 & Mengzi Wang1 & Xi-En Long3 & Guibing Zhu1,2

Received: 16 July 2018 /Accepted: 4 November 2018 /Published online: 15 November 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Purpose In some habitats, ammonia oxidation highly depends on the activity of ammonia-oxidizing archaea (AOA), which are therefore important for studying biogeochemical nitrogen cycling. However, the behavior and distribution of AOA in aquatic ecosystems are not well characterized, especially on a global scale. Materials and methods We sampled 66 sites across all five continents to analyze the global abundance of AOA. Ammonium oxidation rates were measured using the dicyandiamide (DCD) and octyne inhibition method to separately evaluate the contri- butions of AOA to ammonium oxidation. High-throughput pyrosequencing and phylogenetic analysis were applied to study AOA community compositions, combined with DNA-stable isotope probing (DNA-SIP). Results and discussion The archaeal amoA was widespread and abundant across all aquatic types. The average abundance was 3.59 × 108 copies g−1, with the highest values in lake samples and the lowest in river samples. The AOA − −1 −1 abundance was influenced by pH. Archaeal ammonia oxidation rates were 0.81 ± 0.45 mg (NO3 -N) kg day ,corresponding to 63.75% of the total ammonia oxidation rate. Pyrosequencing analysis showed that the AOA community was dominated by the Group I.1b lineage (65.8%). Candidatus Nitrosocosmicus franklandus showed the highest positive correlation with archaeal ammonium oxidation rates and had the highest carbon use efficiency. Conclusions Abundance, activity, and community composition of AOA were highly heterogeneous. pH negatively impacted the abundance of the archaeal amoA functional gene. AOA were the main ammonia oxidators in aquatic ecosystems. Ca. N. franklandus was found to dominate archaeal ammonium oxidation.

Keywords Archaeal ammonium oxidation . Activity . Biogeochemical factors . Nitrosocosmicus . Aquatic ecosystems

1 Introduction environmental problems such as eutrophication. Furthermore, sediments are the main nitrogen sink and repre- Due to the rapid growth of the world’s population, the nitro- sent biocatalytic filters for the water column above (Zhu et al. gen pollution of aquatic ecosystems (which act as nitrogen 2013; Wang et al. 2018a). is the center of the Bsinks^) has increased dramatically, resulting in a series of biogeochemical and determines both the rate and flux of nitrogen removal (Schleper and Nicol 2010; Zhou et al. 2015;Wangetal.2018b). Ammonia oxidation, as the Responsible editor: Terrence H. Bell rate-limiting step of nitrification, has long been assumed to be Electronic supplementary material The online version of this article only conducted by ammonia-oxidizing bacteria (AOB); how- (https://doi.org/10.1007/s11368-018-2188-8) contains supplementary material, which is available to authorized users. ever, the discovery of ammonia-oxidizing archaea (AOA) challenged this assumption (Prosser 1989; Könneke et al. * Guibing Zhu 2005; Stahl and de la Torre 2012). AOA are widely distributed [email protected] throughout aquatic ecosystems, such as marine waters (Francis et al. 2005), inland saline-alkaline wetlands (Gao 1 Research Center for Eco-Environmental Sciences, Chinese Academy et al. 2018), hotsprings (Zhang et al. 2008), constructed wet- of Sciences, Beijing 100085, China lands (Su et al. 2018), and natural freshwater lakes (Liu et al. 2 University of Chinese Academy of Sciences, Beijing 100049, China 2017). As compared to AOB, which dominates nitrification in 3 Key Lab of Urban Environment and Health, Institute of Urban high ammonium nitrogen environments, AOA is known for Environment, Chinese Academy of Sciences, Xiamen 361021, China J Soils Sediments (2019) 19:2114–2125 2115 its higher specific affinity for ammonia and preference to in- environments (Weidler et al. 2008). BCandidatus habit low ammonium nitrogen environments (Martens- Nitrosocosmicus franklandus^ has first been isolated from Habbena et al. 2009; Erguder et al. 2009;Kitsetal.2017) arable soil (pH 7.5) in 2016 and belongs to the and low pH conditions (Nicol et al. 2008). Many previous BNitrososphaera sister cluster^ (Lehtovirta-Morley et al. studies suggested that AOA could outnumber AOB by at least 2016); it is capable of ureolytic growth and has a higher tol- an order of magnitude in nutrient limiting (oligotrophic) erance to and ammonia than other AOA, but a similar aquatic ecosystems, e.g., the Missouri freshwater wetlands, tolerance level compared to typical soil AOB. However, few USA (Sims et al. 2012); Chaohu Lake, China (Wang et al. studies have investigated the importance of Ca. N. franklandus 2018a), and mangrove sediments (Cao et al. 2011). The above in archaeal nitrification (Sauder et al. 2017). Hence, the contri- research results were obtained in a specific environment, and bution of Ca. N. franklandus to ammonia oxidation in aquatic few studies have focused on a wider range. However, the ecosystems should be thoroughly re-assessed. contribution of AOA to ammonia oxidation in a large range In this context, the objectives of this study are to investigate of environments has not been investigated so far (Brochier- the occurrence, distribution, and biodiversity of AOA in Armanet et al. 2008). The discovery of ubiquitous archaeal aquatic ecosystems on a global scale. To achieve this, we used ammonium oxidation greatly stimulated the view of its impor- high-throughput pyrosequencing, quantitative real-time PCR tant role in the global nitrogen cycle and considerable signif- (qPCR), and DNA-stable isotope probing (DNA-SIP) to com- icance in aquatic ecosystems (Liu et al. 2010; Wang et al. pare the abundance, contribution, and possible driver of AOA 2011). communities in various aquatic ecosystems. Ammonia oxidation is catalyzed by ammonia monooxygenase, which is composed of the three subunits amoA, amoB, and amoC. Among them, amoA, encoding 2 Materials and methods the α-subunit of ammonia monooxygenase, is widely used as marker for bacterial and archaeal ammonia oxidizers. 2.1 Study sites Studies have classified AOA as a novel archaeal phylum of the lineage (Brochier-Armanet et al. 2008; We collected and investigated a total of 66 sediment samples Spang et al. 2010), which separates them from the phyla from aquatic ecosystems, including lakes, rivers, paddy fields, Euryarchaeote and . Ammonia-oxidizing ar- reservoirs, and swamps, from eight countries of all five conti- chaea form five distinct phylogenetic clades, namely the nents. Details of the sampling sites, including location, eleva- BNitrosopumilus cluster^ (also called group I.1a), the tion, sediment type, and temperature are shown in Fig. 1 and BNitrososphaera cluster^ (also called group I.1b), the Table S1 (Electronic Supplementary Material (ESM)). Surface BNitrososphaera sister cluster,^ the ‘Nitrosotalea cluster,^ sediment samples (0–10 cm) were collected from each sam- and the BNitrosocaldus cluster (ThAOA)^ (Pester et al. pling site in triplicate, sealed in sterile plastic bags, and imme- 2012). Generally, AOA from the sediments and water of the diately transported on ice to the laboratory. A portion (10 g) of ocean are clustered in group I.1a, while terrestrial AOA are each of the fresh samples was stored at − 80 °C for DNA distributed in the group I.1b branch (Nicol and Schleper extraction and other molecular analyses. A further portion 2006). The cluster ThAOA is distinct from the other branches (100 g) was air-dried and sieved through a 2-mm mesh for and mostly occurs in hot springs and high-temperature physicochemical analyses. The remaining samples were

Fig. 1 Location of the sampling sites. Circles of different colors represent different aquatic ecosystem types, and the number in the circle represents the number of samples. In total, 66 sediment samples were collected from eight countries in five continents 2116 J Soils Sediments (2019) 19:2114–2125 incubated at in situ temperature (9–29 °C) to determine mi- generated by a 10-fold dilution of a plasmid with the amoA crobial activities. gene of a known concentration. The detection limit of the qPCR was 1.00 × 103 copies g−1. The results with an amplifi- 2.2 Analysis of environmental variables cation efficiency and a correlation coefficient (R2) above 95% and 0.98, respectively were employed. All tests were per- + − Ammonium (NH4 ) and nitrite plus nitrate (NOx )wereex- formed in triplicate. tracted from the fresh sediments with 2 M KCl solution and measured using a continuous flow analyzer (SAN plus, Skalar 2.5 Measurement of ammonia-oxidizing activity Analytical B.V. the Netherlands). Total nitrogen (TN), total carbon (TC), total sulfur (TS), and total phosphorus (TP) con- Out of a total of 66 samples, 15 representative samples were tents were measured using an elemental analyzer selected for the determination of the ammonia oxidation rate. (ELEMENTAR, Germany). The pH was determined after These 15 samples covered five continents and three types of mixing with water at a ratio (sediment/water) of 1:5, and total aquatic ecosystems. Laboratory microcosms for sediments organic matter (TOM) was determined via K2Cr2O7 oxidation were performed in 60-mL serum bottles containing 10 g of (Bao 2000). All analyses were performed in triplicate. The sieved fresh samples and were sealed with rubber stoppers and contents were determined per kilogram of dry soil. aluminum caps. Each sample underwent three treatments and three replicates were conducted for each treatment. In the con-

2.3 DNA extraction, PCR, and 454 pyrosequencing trol group, the equivalent volumes of ddH2Owereaddedto bottles to achieve a moisture content approximating the field We extracted DNA from 0.33 g of freeze-dried sediment, capacity of each sample. In addition, two inhibitors were used: using the FastDNA® Spin Kit for Soil (MP Biomedicals, dicyandiamide (DCD) to completely inhibit the ammonium USA), according to the manufacturer’s instructions. oxidation activity of AOA and AOB, and octyne to specifi- Concentrations of the extracted DNA were determined via cally inhibit that of AOB (Zhang et al. 2012; Taylor et al. spectrophotometric analysis in a NanoDrop 200 UV-Vis spec- 2013). In the DCD group, the same volume of DCD solution trophotometer (Thermo Fisher Scientific, USA), and the qual- wasaddedasinddH2O in the control group to obtain ity was checked by electrophoresis on a 1% (weight/vol%) 50 mg DCD kg−1 dry soil. In the octyne group, the same agarose gel. The archaeal amoA (ammonia monooxygenase volume of ddH2O was added as in the control group, and α-subunit) gene was amplified using the barcoded primer 6.8 mL gaseous octyne was injected into the laboratory bottles pairs Archea-amoAF/Archea-amoAR, according to Francis according to the air/water phase partitioning based on Henry’s et al. (2005). All PCR reactions were performed with Ex constant to achieve 4 μM aqueous phase concentrations (Caq) Taq™ polymerase (Takara, Dalian, China). under experimental conditions. Briefly, the samples were pre- The PCR products were multiplex-sequenced on a 454- incubated for 1 day to recover both AOA and AOB activity; pyrosequencing platform (454 FLX+, USA) at Personalbio then, samples were incubated at in situ temperatures (Shanghai, China). High-quality unique DNA sequences were (Table S1—ESM) in the dark. The serum bottles were opened obtained using Quantitative Insights into Microbial Ecology at 0, 5, 10, and 15 days after the beginning of the incubation − (QIIME) (Caporaso et al. 2010) and Mothur (Schloss et al. and 0.5 g sediment samples were extracted for NOx determi- 2009). Operational taxonomic units (OTUs) were clustered at nation. Air was exchanged after bottles were opened to main- 85% similarity level; the coverages exceeded 92% for all sam- tain aerobic conditions. In the octyne group, the original vol- − ples in this study. Phylogenetic trees were constructed in ume of octyne was re-injected. NOx was extracted from all MEGA v6.0 (Tamura et al. 2013). The AOA sequences gen- microcosms with 2 M KCl and determined by a continuous erated in this study were deposited in the GenBank database flow analyzer. under the accession number SRP111034. 2.6 DNA-SIP microcosm and cloning, phylogenetic 2.4 Quantitative real-time PCR analysis analysis

Quantitative real-time PCR (qPCR) was performed on an ABI DNA-SIP microcosm analysis was performed for two typical 7300 real-time PCR instrument (Applied Biosystems, USA) sediment samples under moderate conditions (temperature, with a SYBR Green qPCR kit (Takara, Dalian, China). The ammonia nitrogen content, and nitrite content). Twenty grams qPCR thermal profile of the archaeal amoA gene was obtained fresh sediments were placed into a 125-mL brown serum bot- with the primers Archea-amoAF/Archea-amoAR at an an- tle that was sealed with rubber stoppers and aluminum caps. nealing temperature of 53 °C. Plasmid standards containing Quadruplicate microcosms of each sample were established in 13 the target were generated from appropriate positive this study. Incubation was performed with 5% (v/v)of CO2 12 clones obtained from clone libraries. The standard curves were or CO2 (99 atoms%, Sigma-Aldrich Co., St Louis, MO, J Soils Sediments (2019) 19:2114–2125 2117

USA) at 25 °C in the dark. Microcosms received with fresh air the interquartile range (IQR) were calculated to show the sta- 13 12 and 5% (v/v)of CO2 or CO2 every 2 days to maintain ble range of the abundance of the AOA amoA gene using aerobic conditions. Sediments were sampled after 21 days of SPSS 22.0. Redundancy analysis (RDA) ordination plots incubation. were constructed to determine multivariate relationships be- 12 13 The extracted DNA from C- and C-CO2 incubations tween AOA community compositions and physicochemical was used for density gradient centrifugation and the utilized parameters, using CANOCO 4.5 (Ithaca, NY, USA). First, reagent materials were prepared following Zhang et al. (2012) detrended correspondence analysis (DCA) was conducted to with minor modifications. Briefly, 2.0 μg DNA was mixed determine the unimodality of the taxonomic abundance. The with stock CsCl solution and the initial density was adjusted gradient lengths for the first four axes were 2.427, 2.197, to 1.69 g mL−1 by adding small amounts of TE buffer (0.1 M 2.218, and 2.201, indicating that the AOA community showed Tris-HCl pH 8.0, 0.1 M KCl, and 1.0 mM EDTA) based on the a linear distribution (gradient lengths above 3 indicate infractive index of 1.40 (1.696 g ml−1 buoyant density) read unimodality). Unless otherwise specified, the significance lev- with an AR200 handheld refractometer (Reichert, Inc., el was set at p ≤ 0.05. Graphing was performed using the soft- Buffalo, NY, USA). The mixture was transferred to 4.9-mL ware package Origin 8.0. quick-seal polyallomer tubes (Beckman Coulter).

Ultracentrifugation was performed at 228166 gav (56,200 rpm) for 24 h at 20 °C in a Vti 90 vertical rotor. 3 Results Three hundred milliliters DNA fractions were collected by displacing the CsCl solution with sterile water at the top of 3.1 Environmental conditions of the sampling the tube using a fraction recovery system (Beckman Coulter) habitats and an MPP-100 Mini Peristaltic Pump (CBS Scientific Co., Del Mar, CA, USA). The buoyancy density of each fraction The physicochemical characteristics of sediment samples from was determined by the refractive index reading on the AR200 different aquatic ecosystem types were analyzed (Table 1). handheld refractometer (Reichert, Inc., Buffalo, NY, USA). Sediment pH varied from 4.53 to 8.61; the beach samples were DNAwas precipitated for 2 h at 37 °C by adding two volumes alkaline, while the paddy field samples were slightly acidic. of PEG 6000 in 1.6 M NaCl, then washing with 70% ethanol + The contents of ammonium (NH4 ) in sediments ranged from μ − − twice, and dissolving the DNA pellet in 20 L sterile water. 0.37 to 336.30 mg kg 1 with an average of 87.30 mg kg 1.Lake 13 The amoA genes in the C-DNA were also amplified for and paddy field samples had high ammonium nitrogen loads, 13 clone library construction of the CO2-labeled microcosm while river samples had the lowest levels. The contents of ni- after incubation. A of the amoA genes was − trate and nitrite (NOx ) presented similar patterns as compared also constructed in MEGA v6.0. The cloned AOA sequences + with NH4 -N, with highest values in paddy fields. The contents obtained in this study were deposited in the NCBI under ac- of sedimentary total organic matter (TOM) were in the range of – − cession numbers MF575852 MF576057. 0.02–120.64 g kg 1, and maximum values were found in rivers. Total nitrogen (TN) levels were similar across all samples, with −1 2.7 Data analysis an average value of 2.07 g kg .

Correlations between AOA abundance levels and physico- 3.2 Ubiquity and abundance of AOA in global aquatic chemical characteristics were computed using SPSS 22.0 for ecosystem types Windows (SPSS Inc., USA). Analysis of variance (ANOVA) and paired-sample t tests were conducted to compare the dif- The archaeal amoA gene was detected in all samples, with ferences in the explanatory variables. The median value and abundance values ranging from 104 to 109 copies g−1 (dry

Table 1 Physicochemical characteristics of sediment samples from different wetland types

+ −1 − −1 −1 −1 Wetland types Number Moisture content (%) pH NH4 (mg kg )NOx (mg kg ) TOM (g kg )TN(gkg)

Beach 5 34.58 ± 2.65 7.93 ± 0.48 59.69 ± 44.61 8.45 ± 2.62 8.06 ± 3.23 1.70 ± 0.19 Lake 25 34.71 ± 11.23 7.41 ± 0.99 118.90 ± 99.63 14.17 ± 9.67 22.08 ± 20.65 1.85 ± 1.21 Paddy field 2 24.46 ± 1.21 6.60 ± 0.07 157.92 ± 29.47 24.04 ± 23.38 39.15 ± 7.56 2.64 ± 0.63 Reservoir 2 43.01 ± 3.74 7.79 ± 0.32 93.35 ± 95.86 20.46 ± 19.74 45.50 ± 39.46 1.53 ± 0.61 River 30 33.13 ± 12.33 7.73 ± 0.46 58.17 ± 76.55 6.07 ± 4.71 21.98 ± 27.76 2.35 ± 1.68 Swamp 3 35.95 ± 10.36 6.92 ± 2.07 90.70 ± 77.46 11.17 ± 1.01 8.46 ± 2.09 2.22 ± 0.29 2118 J Soils Sediments (2019) 19:2114–2125 soil), in accordance with the logarithmic normal distribution relationship was found between archaeal ammonium oxida- (Shapiro-Wilk test, p =0.959,n =66;Fig.2). Average abun- tion rates and AOA abundance (p >0.05). dance level of all samples was 3.59 × 108 ±1.22× − 108 copies g 1. Furthermore, more than 81.8% of these abun- 3.4 Diversity and community composition of AOA − dance values ranged from 106 to 108 copies g 1, which was in different aquatic ecosystem types considered to be the widespread abundance level of the ar- chaeal amoA gene in aquatic ecosystems around the world. A total of 66 OTUs, based on 85% similarity (Purkhold et al. − Almost all of the highest values (over 1.00 × 109 copies g 1) 2000; Xia et al. 2011; Pester et al. 2012), were recovered with were detected in lakes, with an average of 8.1 × 108 ±1.89× high diversity in sediment samples (Fig. 4). Pyrosequencing − 108 copies g 1 (n = 25). The river samples showed the lowest analysis of the archaeal amoA gene indicated that the AOA abundance of AOA, and 62.1% of the river samples had an communitywasdominatedbythegroup1.1blineage abundance lower than the average level. All beach and paddy (65.8%), while group1.1a lineage and group I.1a-associated field samples showed a lower abundance than the average lineage only accounted for a small portion (less than 10.0%). level. The swamp samples showed a wider variance (CV = The group 1.1b lineage consisted of three major clusters, 31.6%) than samples from the other types (CV < 15.9%). namely 54d9, Nitrososphaera gargensis,andNitrososphaera Based on the above results, the abundance of AOA showed viennensis, accounting for 29.6, 31.3, and 4.93% of the AOA a spatial heterogeneity at the global scale. It correlated signif- community in the sediments, respectively. Eighteen18 opera- icantly with pH (Pearson’s correlation, r = −0.367, p =0.003, tional taxonomic units were obtained in this study that clus- + Table 2) but did not correlate with substrate NH4 (p =0.369), tered as Ca. N. franklandus, which belonged to the ThAOA − reaction product NO2 (p = 0.658), or other environmental lineage, and accounted for 24.1% in all samples. The compo- variables. In addition, the longitude of sampling sites was a sition of the AOA community at different sampling sites var- further factor that influenced the abundance levels (r = − ied greatly. Specifically, 54d9 was dominant in Tianchi Lake, 0.302, p = 0.014), while latitude and elevation played an in- Simplon Park, and Volcanoland. N. gargensis was the major significant role. taxon in Berryassa Lake, Swan Estuary Marine Park, Nagqu, and Yarlung Zangbo River, respectively. In Nyang River, 3.3 Activities of ammonia oxidizers in different group I.1a lineage and group I.1a-associated lineage were aquatic ecosystem types the dominant lineages. The α-diversity indexes (including Chao1, Ace, Shannon, and Simpson) of each sampling site The measured AOA activity showed that all samples harbored are shown in Table S2 (ESM). High AOA community diver- archaeal ammonium oxidation activities ranging from not de- sity was found in Berryassa Lake, Baiyangdian Lake, Swan − −1 −1 tectable to 4.66 ± 0.02 mg NO3 -N kg dry soil day ,corre- Estuary Marine Park, and the Yarlung Zangbo River. sponding to 0–99.73% of the total ammonia oxidation rate (Fig. 3). The highest value was found in river samples from 3.5 Potential interaction between AOA species the Swan Estuary Marine Park in Australia. The AOA com- and environmental factors munity played the dominant role in nitrification (relative am- monia oxidation rate above 50%) in more than two thirds of Redundancy analysis (RDA) and Pearson’s correlation analy- the samples. The results of the Pearson’s correlation analysis sis were conducted to investigate the potential interaction be- (Table 3) indicated that the archaeal ammonium oxidation tween the relative abundances of AOA species and environ- rates were significantly positively correlated with both TOM mental factors (Fig. 5; Table S3—ESM). The first two RDA (p = 0.016) and latitude (p = 0.02); however, no significant axes explained 50.5 and 17.5% of the variance in the AOA

Fig. 2 Abundance of AOA in different aquatic ecosystem types. Boxes denote interquartile ranges, hollow squares denote mean values, and whiskers denote 1.5 times standard deviations. Solid squares represent the measured values of samples. The blue dotted line indicates the average value of all 66 samples J Soils Sediments (2019) 19:2114–2125 2119

Table 2 Correlation between + − AOA abundance levels and MC TOM pH NH4 NOx TN TP TC physicochemical characteristics Abundance r 0.007 − 0.093 − 0.367** 0.113 − 0.056 − 0.004 − 0.015 − 0.062 or sampling site location (n =66) p 0.955 0.475 0.003** 0.369 0.658 0.979 0.923 0.677 Longitude Latitude Elevation Abundance r − 0.302* − 0.015 − 0.105 p 0.014* 0.902 0.402

**Correlation is significant at the 0.01 level (two-tailed) *Correlation is significant at the 0.05 level (two-tailed) community, respectively, and the species-environmental cor- 13C-Archaeal amoA genes from the heavy fraction were most- relation at the first RDA axis was 99.4%. The relative abun- ly from N. viennensis and N. gargensis, which accounted for dance of Ca. N. franklandus showed the highest positive cor- 47.8% of the fraction, while the other 37.0% were derived relation with archaeal ammonium oxidation rates (r =0.517; from Ca. N. franklandus (Fig. 7). However, in the 12C-labeled p = 0.043). Furthermore, the relative abundance of treatment, only 3.9% of the fraction were from Ca. N. N. gargensis was negatively associated with TOM (p = franklandus and up to 13.7% were from N. gargensis,78.4% 0.026), N. viennensis was positively correlated with the from N. viennensis, and the smallest fraction was mostly from + NH4 level (p = 0.000), and group I.1a lineage and group 54d9-related and OTUs. Therefore, although N. viennensis I.1a-associated lineage were negatively correlated to the pH dominated the archaeal communities in the samples, Ca. N. value of the sediment (p = 0.037; p = 0.021). Therefore, as franklandus had a higher carbon use efficiency and, therefore, compared with other species, Ca. N. franklandus was little a higher growth rate, as combined with a higher potential for impacted by environmental factors. ammonium oxidation.

3.6 Ca. N. franklandus as the key for archaeal ammonium oxidation 4 Discussion

To further substantiate the dominant function of Ca. N. Variations in abundance, activity, and community composi- franklandus, three-peak SIP-DNA fractions were used to con- tion of AOA were explored in different aquatic ecosystem 12 13 struct the clone library for CO2-and CO2-AOA (Fig. 6). types on a global level. The obtained results indicate that the Five species from group I.1b were identified as AOA, based AOA is rich in aquatic ecosystems that exhibited higher bio- on previous species definitions. Phylogenetic investigations, diversity and abundance in the range of 2.48 × 105–4.28 × combined with alignment similarity analysis, suggested that 109 copies g−1. Generally, it is accepted that AOA is

Fig. 3 Ammonium oxidation rates and specific AOA and AOB activities in the representative 15 sediment samples. The gray filling represents the ammonia oxidation rate of the AOA community, while the slash filling represents the ammonia oxidation rate of the AOB community 2120 J Soils Sediments (2019) 19:2114–2125

Table 3 Correlation between AOA activities and physicochemical characteristics or sampling site location (n =15)

+ − MC TOM pH NH4 NOx TN TP TC Activities r − 0.382 −0.339* − 0.026 − 0.018 − 0.177 − 0.272 − 0.049 − 0.290 p 0.160 0.016* 0.928 0.95 0.529 0.326 0.863 0.295 Longitude Latitude Elevation Abundance Activities r 0.413 − 0.594* 0.035 0.718 p 0.126 0.020* 0.902 0.126

**Correlation is significant at the 0.01 level (two-tailed) *Correlation is significant at the 0.05 level (two-tailed) numerically dominant in marine environments (Beman et al. ecosystems, such as polluted wetlands (Wang et al. 2011;Su 2008; Santoro et al. 2010) and soils (Leininger et al. 2006; et al. 2018), wastewater treatment plants (Gao et al. 2013), and Zeglin et al. 2011), while AOB outnumber AOA in aquatic (Wells et al. 2009). However, the existence of

Fig. 4 Results of the phylogenetic analysis of amoA

gene sequences based on 454 River

pyrosequencing. Operational 1 Lake 3 Lake taxonomic units (OTUs) were clustered at 85% similarity level. River NCBI BLAST (http://www.ncbi. Tianchi Lake Baiyangdian Baiyangdian 2 Lake Baiyangdian Park Simplon 1 park marine Swan estuary 2 park marine Swan estuary Nyang Volcanoland Nagqu River Yarlung Zangbo nlm.nih.gov/BLAST)wasusedto Berryassa Lake Weser River River Nampula Island Twitchell Otu403 obtain the closest gene sequences Otu442 Otu142 in the public database. The Otu139 Otu40 number of different colors was Otu39 obtained from the logarithm of the Otu56 54d9 Otu41 number of amoAgenesequences Otu60 Otu24 Otu201 Otu226 Otu224 Otu229 Otu392 Otu397 Otu343 Otu353 Otu137 Otu136 Otu131 Otu185 Otu7 gargensis Otu2 Otu1 Otu278 Otu400 Otu429 Otu344 Otu364 Otu390 Otu133 Otu5 Otu4 Otu37 Nitrososphaera viennensis Otu209 Otu233 Otu234 Otu238 Otu425 Nitrosopumilaceae archaeon Otu377 Otu132 Otu152 Otu204 Nitrosotalea devanaterra Otu326 Otu197 Otu196 Otu309 Otu143 Otu145 Otu138 Otu166 Otu171 Otu50 Otu191 Otu190 Nitrocosmicus franklandus Otu164 Otu27 Otu26 Otu273 Otu252 Otu249 Otu203 Otu202 Otu307 Otu393 J Soils Sediments (2019) 19:2114–2125 2121

+ − abundance level, while NH4 ,NO2 and latitude only played a minor role. This agreed with previous observations (He et al. 2007; Nicol et al. 2008; Erguder et al. 2009). The underlying cause is that the undissociated form of ammonia (rather than + NH4 ) is the actual form of substrate for oxidization by AOB (Suzuki et al. 1974). In contrast, AOA richness responded to + NH4 , which may be directly accessed from the environment by these organisms (Norman and Barrett 2016). Low pH will

reduce the concentration of NH3, which is conducive to the abundant AOA. High-throughput pyrosequencing analysis showed that the AOA community was totally different in sediments and was dominated by the group 1.1b lineage as a whole. Based on previous work, AOA can adapt to a variety of habitats, and AOA species found in one habitat type can also occur in other types (Wang and Gu 2013). Nitrososphaera is the main genus and has, so far, only been isolated from soil and hot springs (Hatzenpichler et al. 2008; Tourna et al. 2011). The group 1.1b lineage microorganisms usually exist in sediment (Liu et al. Fig. 5 Redundancy analysis (RDA) ordination diagram of AOA commu- 2014) and have been referred to as the major AOA compo- nity composition, associated with environmental variable. The percentage nents in lake sediments on the northeastern Qinghai-Tibetan indicated the interpretation of axis to the diversification of thee AOA Plateau (Yang et al. 2013) and the Yunnan Plateau (Liu et al. community 2015) in China. According to the differences in abundance distribution, in different aquatic ecosystems, Nitrososphaera AOA in aquatic ecosystems cannot be ignored. Numerous might contribute to ammonia oxidation to varying degrees. previous studies have shown that environmental variation (in In this study, we determined AOA and AOB activities sep- terms of oxygen concentration, salinity, pH, and ammonia arately and observed a high contribution of archaeal ammonia concentration) most likely affects the abundance and commu- oxidation to nitrification (the AOA ammonia oxidation rate nity composition of AOA (He et al. 2012;ProsserandNicol was 50% higher than that of AOB in two thirds of all sam- 2012;Pett-Ridgeetal.2013). In this study, the pH values and ples). DCD was initially used as nitrification inhibitor in acid longitudinal position were found to highly influence the soils (pH < 4.50) (Zhang et al. 2012). Currently, no consensus has been reached with regard to the stability in alkaline envi- ronments, although it is used in a number of studies (Robinson et al. 2014; Hill et al. 2015; Lan et al. 2018). Lehtovirta- Morley et al. (2013) further indicated several other drawbacks of DCD, e.g., the effect could be reduced in liquid culture, thus requiring repeated application. In this study, the coeffi- cient of determination of the ammonia oxidation rate in sev- eral of the samples was below 0.9, possibly due to the low stability. Therefore, the use of DCD in an alkaline environ- ment will be further discussed. We conclude that AOA most likely play an indispensable role in the nitrification of aquatic ecosystems, considering that AOA occupy a broader habitat range than AOB. However, archaeal ammonium oxidation rates were not significantly related to AOA abundance, sug- gesting that AOA abundance was not the key factor influenc- ing its activity. Similarly, Santoro et al. (2010) also found that Fig. 6 Buoyant density distribution based on 13C-DNA-SIP analysis of two typical sediment samples. Buoyant density distribution of archaeal in water bodies of central California, nitrification rate was not amoA gene abundance in sediment after incubation with a headspace directly related to the abundance of AOA. This variation be- 12 13 concentration of 5% (vol/vol) C- or C-CO2. The values of the tween AOA abundance and activity might be attributed to the plotted points represent the average proportions of total gene abundance diverse physiological features of AOA species, since they throughout the gradient. Vertical and horizontal error bars represent the SE of the proportional abundance and the buoyant density of the fractions possess distinct potentials for ammonium oxidation (Xia from quadruplicate samples, respectively et al. 2011). 2122 J Soils Sediments (2019) 19:2114–2125

74 AOAC12-20 68 AOAC12-61 91 AOAC12-216 96 AOAC12-228 55 AOAC12-198 AOAC12-190 12 87 99 Candidatus Nitrososphaera viennensis EN76 (FR773159) C: 78.4% Candidatus Nitrososphaera evergladensis SR1 (CP007174) 13C: 47.8% AOAC12-1 69

94 viennensis 99 AOAC13-108 64 AOAC13-90 55 AOAC12-116 Nitrososphaera AOAC12-127 Candidatus Nitrososphaera sp. JG1 (JF748723) 95 AOAC12-49 AOAC12-45 94 65 AOAC12-32 AOAC12-16 85 51 AOAC12-35 AOAC12-55 12 AOAC12-40 C: 13.7% AOAC12-5 13C: 13.0%

90 AOAC13-36 gargensis AOAC12-43

100 Nitrososphaera 85 Candidatus (EU281321) Candidatus Nitrososphaera gargensis (EU281320) AOAC13-41 56 AOAC12-94 AOAC12-95 AOAC13-46 12C: 3.9% 68 99 AOAC12-92 AOAC12-93 13C: 0%

AOAC12-90 54d9 AOAC12-75 62 12 51 AOAC12-88 C: 0% 54d9 (AJ627422) 13C: 2.2% 100 hot spring HL4.G09 (EU239971) 96 Candidatus Nitrosocaldus yellowstonii (EU239961) yellowstonii

hot spring NyCr.F07 (EU239978) Nitrosocaldus 85 AOAC13-3 99 AOAC13-2 88 AOAC13-9 12 98 C: 3.9% AOAC12-27 13 99 Candidatus Nitrocosmicus franklandus C13 (KU290366) C: 37.0% 100 Thaumarchaeote enrichment culture clone Ec.MTa2b4-19 (JX426593) Candidatus Nitrocosmicus oleophilus strain:MY3 (CP012850) franklandus Nitrocosmicus 100 AOAC12-98 62 AOAC12-97 99 Group I.1a

0.05 Fig. 7 Phylogenetic analysis of amoA gene sequences in wetlands based reference sequence. Evolutionary distance dendrograms and branching on DNA-SIP analysis. An alignment of the sequences from OTUs and patterns (> 50% values) were constructed using the neighbor-joining reference sequences from the NCBI database were constructed phyloge- method, with the Kimura two-parameter distance with 1000 bootstraps netic trees. GenBank accession numbers are shown following each

Interestingly, the less abundant Ca. N. franklandus showed franklandus. DNA-SIP analysis was a reliable approach to the highest positive correlation with archaeal ammonium ox- identify microbial activity (Pester et al. 2010;Chenand idation rates. Based on a previous study, Ca. N. franklandus is Murrell 2010; Aanderud et al. 2015). In our study, DNA-SIP capable of ureolytic growth and has a higher tolerance to ni- analysis indicated that Ca. N. franklandus plays a major role in trite and ammonia compared to other AOA (< 50 mM), with archaeal ammonia oxidation. In the last 10 years, this AOA similar tolerance levels as those of typical soil AOB (> cluster was sketchily classified as the Nitrososphaera sister 100 mM) (Martens-Habbena et al. 2009; Lehtovirta-Morley cluster, Thaumarchaeota AOA, or even group 1.1b. Only re- et al. 2016). The similarity of growth characteristics of Ca. N. cently has Ca. N. franklandus been isolated from an arable soil franklandus with those of typical soil AOB does not support (pH 7.5) by Lehtovirta-Morley et al. (2016); the author sug- the theory of niche differentiation between AOA and AOB, gested that this taxon greatly contributes to nitrification in but rather suggests that AOA have a wider physiological di- fertilized soils, and the physiological diversity may be more versity than previously suspected. However, it may also indi- important for determining the composition of natural commu- cate that the key to the function of the AOA community is nities. Except for the high tolerance towards ammonium con- probably not the abundance of dominant taxa such as centrations, numerous definite physiological and ecological Nitrososphaera, but other AOA taxa, such as Ca. N. characteristics of Ca. N. franklandus still remain unknown. J Soils Sediments (2019) 19:2114–2125 2123

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